The invention relates to methods for measuring the activity of FlaA1, WbpM and homologues thereof and methods for the isolation and of inhibitors of FlaA1 and WbpM and homologues thereof which are useful as antimicrobial agents.
The opportunistic pathogen P. aeruginosa remains a problem in the nosocomial infection of immunocompromised individuals. P. aeruginosa infections are particularly a problem in burn patients, people receiving medical implants, and in individual suffering from cystic fibrosis (Fick, R. B. Jr., 1993). The organism is intrinsically resistant to many antibiotics and capable of forming biofilms which are recalcitrant to treatment. Several virulence factors have been identified in the pathogenesis of P. aeruginosa infections, including proteins such as exotoxin A, proteases, and exopolysaccharides including alginate and lipopolysaccharide (LPS). The LPS of P. aeruginosa is typical of Gram-negative bacteria, composed of lipid A-core oligosaccharide-O antigen repeating units.
P. aeruginosa is capable of coexpressing two distinct forms of LPS, designated A-band and B-band LPS, respectively. A-band LPS is a shorter, common form expressed by the majority of P. aeruginosa serotypes, and has a trisaccharide repeating unit of xcex1-D-rhamnose linked 1xe2x86x923, 1xe2x86x923, 1xe2x86x922. B-band LPS is the serotype-specific, O-antigen-containing form, and is a heteropolymer composed of di- to pentasaccharide repeats containing a wide variety of acyl sugars, amino sugars, and uronic acids. Both the A- and B-band repeating units are attached to lipid A-core, but there appears to be differences between them regarding point of attachment to and composition of the outer core region (Rivera et al., 1992).
The gene clusters for biosynthesis of core oligosaccharides/O-antigens rfb have been cloned and characterized from several bacterial species, including some from non-enteric genera such as Bordetella (Allen and Maskell, 1996), Haemophilus (Jarosik and Hansen, 1994), Neisseria (Gotschlich, 1994), Vibrio (Stroeher et al., 1992; Amor and Mutharia, 1995; Comstock et al., 1996), and Xanthamonas (Kingsley et al., 1993).
The clusters of genes involved in the biosynthesis of the O-antigen for serotype O5 and O6 of Pseudomonas aeruginosa have recently been sequenced (Burrows et al. 1996; Belanger et al. 1999). Based on sequence homologies, putative enzymatic functions have been assigned to the various gene products, and pathways have been proposed for the synthesis of specific sugar derivatives that constitute the O-antigen tri-saccharide repeats. WbpM is an essential gene required for expression of B-band LPS in both serotypes. The WbpM gene is conserved among all 20 serotypes of P. aeruginosa, and homologues have also been identified in a large number of bacterial pathogens including Helicobacter pylori (Allen and Maskell (1996); Lin W. S. et al. (1994); Comstock L. E. et al. (1996); Zhang, L. et al. (1997); Tomb, J.-F. et al. (1997)).
WbpM is a large protein of 665 amino acids (75 kDa). The C-terminus portion of the protein presents 4 domains that are conserved among all WbpM homologues. The precise function of the encoded protein WbpM is not known, but knock-out experiments have shown that it is essential for B-band LPS biosynthesis in several serotypes, including serotype O5. Sequence analysis suggests that it might be involved in the biosynthesis of UDP-N-acetylfucosamine. Prior to the present invention it was unknown whether WbpM was a C4 epimerase catalyzing the conversion of UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine, or if it was a dehydratase involved in the conversion of UDP-N-acetylgalactosamine to UDP-N-acetylfucosamine.
Helicobacter pylori (H. plori) is a microaerophilic Gram-negative bacteria that has been associated with gastric diseases such as ulcers and cancers (Warren and Marshall 1983, Graham 1991, Peterson 1991). It is present chronically in 70-90% of the population in developing countries (Dunn et al. 1997) but its mode of transmission and its potential initial reservoir are not known. This organism is well adapted to the hostile environment in which it thrives, thanks to the production of high level of urease to neutralize acidic pH (Smoot et al. 1990). Its virulence has been associated with its motility that is conferred by a unipolar sheated flagella (Josenhans et al. 1995, Eaton et al. 1992), with its capacity to create acidic vacuoles in epithelial cells (Labigne and De Reuse 1996), as well as with its lipopolysaccharide (LPS) (Muotiala et al. 1992). The LPS of H. pylori exhibits structural features also found at the surface of human blood cells such as the presence of Lewis X and Y antigenic determinants (Aspinall and Monteiro 1996; Aspinall et al. 1996). Such structures are thought to play a role as molecular mimics that allow the organism to evade host immunity defences (Sherburne and Taylor 1995).
The entire genomes of H. pylori strains 26695 and J99 have been sequenced recently (Tomb et al. 1997; Alm et al. 1999). Though the genomes are quite small compared with that of other Gram-negative bacteria, numerous open reading frames (ORF) could not be assigned a putative function based on sequence homologies. This is mainly due to the fact that, contrarily to other well studied gram-negative bacteria, the genome of H. pylori is not organised into operons which regroup genes involved in the same biological functions into defined clusters. In cases where only low sequence homologies with genes from other organisms are observed, the gene position is not useful to assign a putative function. FlaA1 (HP0840) is one of these ORFs found in strain 26695. It was originally assigned a flagellar-related function, hence its name, though it is not linked to any other flagellar biosynthetic and/or assembly genes. The original assignment is consistent with the existence of two homologues of FlaA1 found in Caulobacter crescentus (FlmA, Leclerc et al. 1998) and in Campylobacter jejuni (PglF, Szymanski et al. 1999). Both have been shown to be involved in glycosylation of flagella proteins by knock-out analysis. However, no biochemical evidence is available to assign a specific enzymatic function to the gene products. Moreover, glycosylation of flagella has not been demonstrated in H. pylori itself, although it has been demonstrated in several species of Campylobacter (Doig et al. 1996, Szymanski et al. 1999), which is closely related to H. pylori. 
As the FlaA1 or WbpM enzymes are not present in higher organisms (such as mammals) they represent good drug target candidates for the development of new antimicrobial agents that would be efficient against H. pylori or P. aeruginosa as well as other organisms that produce homologues of FlaA1 or WbpM. However, before new antimicrobial agents can be developed, the biochemical pathways that are catalysed by the enzymes must be determined.
The present inventors have determined that a series of homologous enzymes from a variety of human bacterial pathogens are C6 sugar-nucleotide dehydratases. In particular, the inventors have shown that FlaA1 from Helicobacter pylori, WbpM from Pseudomonas aeruginosa, BplL from Bordetella pertussis, Cap8D from Staphylococcus aureus and TrsG from Yersinia enterocolitica are C6-dehydratases which allows the development of biochemical assays to detect these enzymes as well as the isolation of agents that inhibit these enzymes. Agents that inhibit the enzymes can be used to combat the bacterial pathogens containing the enzymes.
In one aspect, the present invention provides an assay for measuring the activity of or detecting FlaA1, WbpM and functional homologues thereof. In particular, the present invention provides a spectrophotometric assay for measurement of the activity of FlaA1, WbpM or a homologue thereof that is substrate/product specific.
Accordingly, the present invention provides a method for detecting an enzyme with FlaA1-like activity in a sample comprising the steps of: (a) incubating the sample with UDP-GlcNAc; (b) stopping the reaction; and (c) determining if there has been a decrease in UDP-GlcNAc in the sample, wherein a decrease in UDP-GlcNAc indicates the presence of an enzyme with FlaA1-like activity.
In another aspect, the present invention provides an assay for detecting inhibitors of FlaA1, WbpM or a homologue thereof. Accordingly, the present invention further provides a method for screening for an inhibitor of an enzyme with FlaA1-like activity comprising (a) incubating a test sample containing (i) an enzyme with FlaA1-like activity, (ii) a substance suspected of being an inhibitor of the enzyme; and (iii) UDP-GlcNAc; (b) stopping the reaction; (c) comparing the amount of UDP-GlcNAc in the test sample with the amount in a control sample (that does not contain the substance suspected of being an inhibitor) wherein a decrease in the amount of GlcNAc in the control sample as compared to the test sample indicates that the substance is an inhibitor of the enzyme.
In a further aspect, the present invention provides a method for preparing UDP-N-acetyl quinovosamine (UDP-QuiNAc) using the FlaA1 enzyme or a homologue thereof and UDP-GlcNAc as a substrate.